EP0674712B1 - A proteinase inhibitor, precursor thereof and genetic sequences encoding same - Google Patents

A proteinase inhibitor, precursor thereof and genetic sequences encoding same Download PDF

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Publication number
EP0674712B1
EP0674712B1 EP94902551A EP94902551A EP0674712B1 EP 0674712 B1 EP0674712 B1 EP 0674712B1 EP 94902551 A EP94902551 A EP 94902551A EP 94902551 A EP94902551 A EP 94902551A EP 0674712 B1 EP0674712 B1 EP 0674712B1
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precursor
seq
sequence
nucleic acid
plant
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EP0674712A4 (en
EP0674712A1 (en
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Marilyn Anne Anderson
Angela Hilary Atkinson
Robyn Louise Heath
Adrienne Elizabeth Clarke
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Hexima Ltd
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Hexima Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/06—Uses of viruses as vector in vitro
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/81—Protease inhibitors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • the present invention relates generally to proteinase inhibitors, a precursor thereof and to genetic sequences encoding same.
  • SEQ ID NOs Nucleotide and amino acid sequences are referred to herein by sequence identity numbers (SEQ ID NOs) which are defined after the bibliography. A general summary of the SEQ ID NOs is provided before the examples.
  • the inhibitors can comprise 10% or more of the stored proteins (Richardson, 1977), while in leaves of tomato and potato (Green and Ryan, 1972), and alfalfa (Brown and Ryan, 1984), proteinase inhibitors can accumulate to levels of 2% of the soluble protein within 48 hours of insect attack, or other types of wounding (Brown & Ryan, 1984; Graham et al ., 1986). High levels of these inhibitors (up to 50% of total soluble protein) are also present in unripe fruits of the wild tomato, Lycopersicon peruvianum (Pearce et al ., 1988).
  • Type I inhibitors are small proteins (monomer Mr 8100) which inhibit chymotrypsin at a single reactive site (Melville and Ryan, 1970; Plunkett et al ., 1982).
  • Inhibitors of the type II family generally contain two reactive sites, one of which inhibits chymotrypsin and the other trypsin (Bryant et al ., 1976; Plunkett et al ., 1982).
  • the type II inhibitors have a monomer Mr of 12,300 (Plunkett et al ., 1982).
  • Proteinase inhibitor I accumulates in etiolated tobacco ( Nicotiana tabacum ) leaves (Kuo et al ., 1984), and elicitors from Phytophthora parasitica var. nicotianae were found to induce proteinase inhibitor I accumulation in tobacco cell suspension cultures (Rickauer et al ., 1989).
  • genetic sequences encoding a proteinase inhibitor precursor have been cloned.
  • the precursor has multi-proteinase inhibitor domains and will be useful in developing a range of transgenic plants with enhanced proteinase inhibitor expression. Such plants will have enhanced protective properties against pathogens and predators.
  • the genetic constructs of the present invention will also be useful in developing vaccines for ingestion by insects which are themselves predators or which act as hosts for plant pathogens.
  • the recombinant precursor or monomeric inhibitors will also be useful in topical sprays and in assisting animals in feed digestion.
  • one aspect of the present invention relates to a nucleic acid molecule comprising a sequence of nucleotides which encodes or is complementary to a sequence which encodes a type II serine proteinase inhibitor (PI) precursor from a plant wherein said precursor comprises at least three PI monomers and wherein at least one of said monomers has a chymotrypsin specific site and at least one other of said monomers has a trypsin specific site.
  • PI serine proteinase inhibitor
  • nucleic acid molecule of the present invention may be RNA or DNA (eg cDNA), single or double stranded and linear or covalently closed.
  • the nucleic acid molecule may also be genomic DNA corresponding to the entire gene or a substantial portion thereof or to fragments or derivatives thereof.
  • the nucleotide sequence may correspond to the naturally occurring nucleotide sequence of the genomic or cDNA clone or may contain single or multiple nucleotide substitutions, deletions and/or additions thereto. All such variants in the nucleic acid molecule either retain the ability to encode at least one monomer or active part thereof or are useful as hybridisation probes or polymerase chain reaction (PCR) primers for the same or similar genetic sequences in other sources.
  • PCR polymerase chain reaction
  • the PI precursor comprises at least four, more preferably at least five and even more preferably at least six PI monomers. Still more preferably, the PI precursor further comprises a signal sequence.
  • the PI precursor of the present invention preferably comprises amino acid sequences which are process sites for cleavage into individual monomers.
  • precursor as used herein is not intended to place any limitation on the utility of the precursor molecule itself or a requirement that the molecule first be processed into monomers before PI activity is expressed.
  • the precursor molecule has PI activity and the present invention is directed to the precursor and to the individual monomers of the precursor.
  • the present invention extends to a nucleic acid molecule comprising a sequence of nucleotides which encodes or is complementary to a sequence which encodes a hybrid type II serine PI precursor wherein said precursor comprises at least two monomers from different PIs.
  • the at least two monomers may be modified such as being unable to be processed into individual monomers or may retain the ability to be so processed.
  • at least one of said monomers has a chymotrypsin specific site and the other of said monomers has a trypsin specific site.
  • At least one of said monomers is a thionin.
  • Such hybrid PI precursors and/or monomers thereof are particularly useful in generating molecules which are "multi-valent” in that they are active against a range of pathogens and predators such as both fungi and insects. Accordingly, reference herein to "PI precursor” includes reference to hybrid molecules.
  • the present invention is exemplified by the isolation of the subject nucleic acid molecule from Nicotiana alata which has the following nucleotide sequence (SEQ ID NO. 1) and a corresponding amino acid sequence (SEQ ID NO. 3):
  • nucleic acid molecule from any other plant.
  • equivalent and substantially similar is meant at the level of nucleotide sequence, amino acid sequence, antibody reactivity, monomer composition and/or processing of the precursor to produce monomers.
  • a nucleotide sequence having a percentage sequence similarity of at least 55%, such as about 60-65%, 70-75%, 80-85% and over 90% when compared to the sequence of SEQ ID NO. 1 would be considered “substantially similar” to the subject nucleic acid molecule provided that such a substantially similar sequence encodes a PI precursor having at least three monomers and preferably four, five or six monomers as hereinbefore described.
  • the nucleic acid molecule further encodes a signal sequence 5' to the open reading frame and/or a nucleotide sequence 3' of the coding region providing a full nucleotide sequence as follows (SEQ ID NO. 2): including substantially similar variants thereof.
  • a preferred embodiment of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides as set forth in SEQ ID NO. 1 or 2 which encodes or is complementary to a sequence which encodes a type II serine PI precursor from Nicotiana alata or having at least 55% similarity to said precursor or at least one domain therein wherein said precursor comprises a signal peptide and at least five monomers and wherein one of said monomers has a chymotrypsin specific site and four of said monomers have trypsin specific sites.
  • the nucleic acid molecule is a cDNA molecule and comprises a nucleotide sequence generally as set forth in SEQ ID NO. 1 or 2 or being substantially similar thereto as hereinbefore defined to the whole of said sequence or to a domain thereof.
  • nucleic acid molecule comprising a sequence of nucleotides which encodes or is complementary to a sequence which encodes a single type II serine PI having either a chymotrypsin specific site or a trypsin specific site and wherein said PI is a monomer of a precursor PI having at least three monomers of which at least one of said monomers has a chymotrypsin site and the other of said monomers has a trypsin site.
  • the precursor has four, five or six monomers and is as hereinbefore defined.
  • the plant is N. alata (Link et Otto) having self-incompatibility genotype S 1 S 3 , S 3 S 3 or S 6 S 6 , and the nucleic acid molecule is isolatable from or complementary to genetic sequences isolatable from stigmas and styles of mature plants.
  • the corresponding mRNA is approximately 1.4 kb and the cDNA has six conserved domains wherein the first two domains are 100% identical and contain chymotrypsin-specific sites (Leu-Asn). The third, fourth and fifth domains share 95-98% identity and have sites specific for trypsin (Arg-Asn).
  • a sixth domain which also has a trypsin specific site has less identity to the third, fourth and fifth domains (79-90%) due mainly to a divergent 3' sequence (see Table 1).
  • the preferred PI inhibitor of the present invention has a molecular weight of approximately 42-45 kDa with an approximately 29 amino acid signal sequence.
  • the N-terminal sequence of the monomeric PI is represented in each of the six repeated domains in the predicted sequence of the PI precursor protein. Thus, it is likely that the PI precursor protein is cleaved at six sites to produce seven peptides. Six of the seven peptides, peptides 2, 3, 4, 5, 6 and 7 ( Figure 1, residues 25-82 [SEQ ID NO. 5], 83-140 [SEO ID NO. 6], 141-198 [SEQ ID NO. 7], 199-256 [SEQ ID NO. 8], 257-314 [SEQ ID NO. 9] and 315-368 [SEQ ID NO.
  • Peptide 7 does not contain a consensus site for trypsin or chymotrypsin.
  • Peptide 1 (residues 1-24 [SEQ ID NO. 4], Figure 1) is smaller than 6 kD, has a different N-terminus and was not detected in a purified monomeric PI preparation. It could be envisaged that peptide 1 and peptide 7 would form a functional proteinase inhibitor with the inhibitory site on peptide 1 held in the correct conformation by disulphide bonds formed between the two peptides.
  • the PI precursor may be processed by a protease responsible, for example, for cleavage of an Asn-Asp linkage, to produce the bioactive monomers.
  • the protease sensitive sequence is R 1 -X 1 -X 2 -Asn-Asp-R 2 where R 1 , R 2 , X 1 and X 2 are defined below. The discovery of such a sequence will enable the engineering of peptides and polypeptides capable of being processed in a plant by cleavage of the protease sensitive sequence.
  • protease sensitive peptide comprising the amino acid sequence: wherein X 1 and X 2 are any amino acid but are preferably both Lys residues.
  • the protease sensitive peptide may also be represented as: wherein X 1 and X 2 are preferably the same and are preferably both Lys residues and wherein R 1 and R 2 are the same or different, any D or L amino acid, a peptide, a polypeptide, a protein, or a non-amino acid moiety or molecule such as, but not limited to, an alkyl (eg methyl, ethyl), substituted alkyl, alkenyl, substituted alkenyl, acyl, dienyl, arylalkyl, arylalkenyl, aryl, substituted aryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, halo (e.g.
  • alkyl, substitued alkyl, alkenyl and substituted alkenyl and the like is meant to encompass straight and branched molecules, lower (C 1 - C 6 ) and higher (more than C 6 ) derivatives.
  • substituted includes all the substituents set forth above.
  • the protease sensitive peptide is: wherein R 1 and R 2 are the same or different and are peptides or polypeptides and wherein X 1 and X 2 are both Lys residues.
  • protease sensitive peptide can be placed between the same or different monomers so that upon expression in a suitable host or in vitro , the larger molecule can be processed to the peptides located between the protease sensitive peptides.
  • the present invention also extends to a nucleic acid molecule comprising a sequence of nucleotides which encodes or is complementary to a sequence which encodes a protease sensitive peptide comprising the sequence: wherein X 1 and X 2 are preferably the same and are most preferably both Lys residues.
  • a nucleic acid molecule may be part of a larger nucleotide sequence encoding, for example, a precursor polypeptide capable of being processed via the protease sensitive sequence into individual peptides or monomers.
  • protease sensitive peptide of the present invention is particularly useful in generating poly and/or multi-valent "precursors" wherein each monomer is the same or different and directed to the same or different activities such as anti-viral, antibacterial, anti-fungal, anti-pathogen and/or anti-predator activity.
  • protease acts adjacent the Asn residue as more particularly between the Asn-Asp residues.
  • the present invention extends to an isolated type II serine PI precursor from a plant wherein said precursor comprises at least three PI monomers and wherein at least one of said monomers has a chymotrypsin specific site and at least one other of said monomers has a trypsin specific site.
  • the PI precursor has four, five or six monomers and is encoded by the nucleic acid molecule as hereinbefore described.
  • the present invention also extends to the individual monomers comprising the precursor.
  • the present invention also extends to a hybrid recombinant PI precursor molecule comprising at least two monomers from different PIs as hereinbefore described.
  • the isolated PI or PI precursor may be in recombinant form and/or biologically pure.
  • biologically pure is meant a preparation of PI, PI precursor and/or any mixtures thereof having undergone at least one purification step including ammonium sulphate precipitation, Sephadex chromatography and/or affinity chromatography.
  • the preparation comprises at least 20% of the PI, PI precursor or mixture thereof as determined by weight, activity antibody, reactivity and/or amino acid content. Even more preferably, the preparation comprises 30-40%, 50-60% or at least 80-90% of PI, PI precursor or mixture thereof.
  • the PI or its precursor may be naturally occurring or be a variant as encoded by the nucleic acid variants referred to above. It may also contain single or multiple substitutions, deletions and/or additions to its amino acid sequence or to non-proteinaceous components such as carbohydrate and/or lipid moieties.
  • the recombinant and isolated PI, PI precursor and mixtures thereof are useful as laboratory reagents, in the generation of antibodies, in topically applied insecticides as well as orally ingested insecticides.
  • the recombinant PI or PI precursor may be provided as an insecticide alone or in combination with one or more carriers or other insecticides such as the BT crystal protein.
  • the PI of the present invention is considered to have a defensive role in organs of the plant, for example, the stigma, against the growth or infection by pests and pathogens such as fungi, bacteria and insects.
  • a defensive role in organs of the plant for example, the stigma
  • pests and pathogens such as fungi, bacteria and insects.
  • another aspect of the present invention contemplates a genetic construct comprising a nucleic acid molecule comprising a sequence of nucleotides which encodes or is complementary to a sequence which encodes a type II serine PI precursor or monomer thereof from a plant wherein said precursor comprises at least three PI monomers and wherein at least one of said monomers has a chymotrypsin specific site and at least one of said other monomers has a trypsin specific site and said genetic sequence further comprises expression means to permit expression of said nucleic acid molecule, replication means to permit replication in a plant cell or, alternatively, integration means, to permit stable integration of said nucleic acid molecule into a plant cell genome.
  • the expression is regulated such as developmentally or in response to infection such as being regulated by an existing PI regulatory sequence.
  • the expression of the nucleic acid molecule is enhanced to thereby provide greater endogenous levels of PI relative to the levels in the naturally occurring plant.
  • the PI precursor cDNA of the present invention is usable to obtain a promoter sequence which can then be used in the genetic construct or to cause its manipulation to thereby permit over-expression of the equivalent endogenous promoter.
  • the PI precursor is a hybrid molecule as hereinbefore described.
  • Yet another aspect of the present invention is directed to a transgenic plant carrying the genetic sequence and/or nucleic acid molecule as hereinbefore described and capable of producing elevated, enhanced or more rapidly produced levels of PI and/or PI precursor or hybrid PI precursor when required.
  • the plant is a crop plant or a tobacco plant but other plants are usable where the PI or PI precursor nucleic acid molecule is expressable in said plant.
  • the transgenic plant produces PI precursor, the plant may or may not further process the precursor into monomers.
  • the genetic sequence may be part of a viral or bacterial vector for transmission to an insect to thereby control pathogens in insects which would consequently interrupt the transmission of the pathogens to plants.
  • antibodies to the PI precursor or one or more of its monomers may be monoclonal or polyclonal and are useful in screening for PI or PI precursor clones in an expression library or for purifying PI or PI precursor in a fermentation fluid, supernatant fluid or plant extract.
  • the genetic constructs of the present invention can also be used to populate the gut of insects to act against the insect itself or any plant pathogens therein or to incorporate into the gut of animals to facilitate the digestion of plant material.
  • Figure 1 shows the nucleic acid sequence (SEQ ID NO. 2) of the pNA-PI-2 insert and the corresponding amino acid sequence (SEQ ID NO. 3) of the N. alata PI protein.
  • the amino acid sequence is numbered beginning with 1 for the first amino acid of the mature protein.
  • the signal sequence is encoded by nucleotides 1 to 97 and the amino acid residues have been assigned negative numbers.
  • the reactive site residues of the inhibitor are boxed.
  • the N. alata PI sequence contains six similar domains (domain 1, residues 1 to 58, domain 2, residues 59-116, domain 3, residues 117-174, domain 4, residues 175-232, domain 5, residues 233-290 and domain 6, residues 291-343).
  • Figure 2 is a photographic representation showing a gel blot analysis of RNA from various organs of N. alata .
  • Gel Blot of RNA isolated from organs of N. alata and from stigmas and styles of N. tabacum and N. sylvestris hybridised with the cDNA clone NA-PI-2.
  • St stigma and style
  • Ov ovaries
  • Po pollen
  • Pe petals
  • Se sepals
  • L non-wounded leaves
  • L24 leaves 24h after wounding
  • Ns, N. sylvestris stigma and style Na Hin dIII restriction fragments of Lambda-DNA.
  • the NA-PI-2 clone hybridised to 2 mRNA species (1.0 and 1.4kb). The larger mRNA was predominant in stigma and styles, whereas the smaller mRNA species was more dominant in other tissues. After high stringency washes, the 1.0kb mRNA from stigma and style no longer hybridises to the NA-PI-2 probe.
  • Figure 3 is a photographic presentation depicting in situ localisation of RNA homologous to NA-PI-2 in stigma and style.
  • Figure 4 is a photographic representation of a gel blot analysis of genomic DNA of N. alata .
  • Size markers (kb) are Hin dIII restriction fragments of Lambda-DNA.
  • the NA-PI-2 clone appears to belong to a small multigene family consisting of at least two members.
  • Figure 5 is a graphic representation of PI activity in various organs of N. alata . Buffer soluble extracts from various organs were tested for their ability to inhibit trypsin and chymotrypsin. Stigma and sepal extracts were the most effective inhibitors of both trypsin (A) and chymotrypsin (B).
  • Figure 6 depicts the steps of the purification of PI from N. alata stigmas.
  • the PI is a 6kD protein and is a major component in unfractionated buffer soluble extracts from stigmas.
  • Figure 7 is a graphical representation showing hydropathy plots of the PI proteins encoded by the NA-PI-2 clone from N. alata and the potato and tomato PI II cDNAs. Values above the line denote hydrophobic regions and values below the line denote hydrophilic regions. The putative signal peptides are shaded. The hydrophobicity profile was generated using the predictive rules of Kyte and Doolittle (1982) and a span of 9 consecutive amino acids.
  • Figure 8 shows an immunoblot analysis of the PI protein in stigmas of developing flowers.
  • Stigmas from developing flowers contain four proteins of approximately 42kD, 32kD, 18kD and 6kD that bind to the anti-PI antibody.
  • the 42kD and the 18kD components decrease in concentration as the flowers mature, while the 6kD PI protein reaches a maximum concentration just before anthesis.
  • the level of the 32kD component, which runs as a doublet, does not alter significantly during flower development.
  • FIG. 9 shows the separation and identification of the 6kD proteinase inhibitor species from N.alata stigmas
  • Figure 10 shows the amino-acid sequence around the processing sites in the precursor PI protein.
  • the sequence in bold is the amino-terminal sequence obtained from the purified PI protein.
  • the sequence labelled with negative numbers is the flanking sequence predicted from the cDNA clone.
  • the predicted precursor protein contains six repeats of this sequence.
  • Figure 11 shows the PI precursor produced in a baculovirus expression system and the products obtained after digestion of the affinity purified PI precursor by the endoproteinase Asp-N.
  • Figure 12 Preparative chromatography by reversed phase HPLC of the peptides produced from the precursor by Asp-N digestion
  • Figure 13 shows a comparison of the trypsin and chymotrypsin inhibition activity of the PI precursor, PI peptides from stigmas and in vitro produced PI peptides from the PI precursor.
  • PI precursor or PI peptides (0-1.0 ⁇ g) were tested for their ability to inhibit 1.0 ⁇ g of trypsin or chymotrypsin as described in the materials and methods.
  • Inhibitory activity is expressed as the percentage of proteinase activity remaining after the proteinase had been preincubated with the PI with 100% remaining activity taken as the activity of the proteinase preincubated with no PI. Experiments were performed in duplicate and mean values were plotted. Deviation from the mean was 8% or less.
  • Figure 14 is a graphical representation showing a growth curve for T. commodus nymphs reared on control artificial diet, soybean Bowman-Birk inhibitor and N. alata PI.
  • the vertical axis represents the mean weight of the crickets in each treatment (+/- standard error) in mg.
  • the horizontal axis represents the week number.
  • the crickets reared on the N. alata PI showed a lower mean weight than those reared on both the control diet and the diet containing the soybean inhibitor, throughout the experiment.
  • SUMMARY OF SEQ ID NOs SEQ ID NO. 1 Nucleotide coding region of N. alata PI precursor SEQ ID NO. 2 Full length nucleotide sequence of N. alata PI precursor SEQ ID NO.
  • SEQ ID NO. 4 Residues 1-24 of SEQ ID NO. 2 (peptide 1) SEQ ID NO. 5 Residues 25-82 of SEQ ID NO. 2 (peptide 2) SEQ ID NO. 6 Residues 83-140 of SEQ ID NO. 2 (peptide 3) SEQ ID NO. 7 Residues 141-198 of SEQ ID NO. 2 (peptide 4) SEQ ID NO. 8 Residues 199-256 of SEQ ID NO. 2 (peptide 5) SEQ ID NO. 9 Residues 257-314 of SEQ ID NO. 2 (peptide 6) SEQ ID NO. 10 Residues 315-368 of SEQ ID NO. 2 (peptide 7) SEQ ID NO. 11 N-terminal amino acid sequence of 6kD PI protein SEQ ID NO. 12 N-terminal amino acid sequence of 6kD PI protein
  • Nicotiana alata (Link et Otto) plants of self-incompatibility genotype S 1 S 3 , S 3 S 3 and S 6 S 6 were maintained under standard glasshouse conditions as previously described (Anderson et al ., 1989). Organs were collected directly into liquid Nitrogen to avoid induction of a wound response and stored at -70° until required. To study the effect of wounding on gene expression, leaves were wounded by crushing across the midvein with a dialysis clip. Leaves were collected 4 and 24 hours after wounding.
  • Polyadenylated RNA was prepared from stigmas and styles, isolated from mature flowers of N. alata (genotype S 3 S 3 ), and used to construct a cDNA library in Lambda gt10 (Anderson et al., 1989).
  • Single stranded 32 P-labelled cDNA was prepared from mRNA from stigmas and styles of N. alata (genotype S 3 S 3 and S 6 S 6 ) and used to screen the library for highly expressed clones which were not S-genotype specific (Anderson et al ., 1989). Plaques which hybridised strongly to cDNA probes from both S-genotypes were selected and assembled into groups on the basis of cross-hybridisation.
  • sequencing primers were made to non-repeated 3' sequences (nucleotides 1117-1137, 1188-1203 and 1247-1267), and to a 5' sequence before the start of the repetitive regions (nucleotides 74-98).
  • the pNA-PI-2 insert was restricted with endonuclease Hae III, which cut at nucleotides 622 and 970 to produce three fragments. The fragments were subcloned into pGEM7zf + and sequenced in both directions, using the M13 forward and reverse primers.
  • the repetitive nature of the pNA-PI-2 insert rendered it unstable in both phagemid and plasmid vectors when cultures were grown longer than 6 hours.
  • In situ hybridisation was performed as described by Cornish et al ., 1987.
  • the probe was prepared by labelling the insert from pNA-PI-2 (100ng) to a specific activity of 10 8 cpm ⁇ g -1 by random hexanucleotide priming (Feinberg and Vogelstein, 1983).
  • the labelled probe was precipitated, and resuspended in hybridisation buffer (50 ⁇ l), and 5 ⁇ l was applied to the sections.
  • the sections were covered with coverslips, and incubated overnight at 40 °C in a closed box containing 50% v/v formamide.
  • Genomic DNA was isolated from young leaves of N. alata by the procedure of Bematzky and Tanksley (1986). DNA (10 ⁇ g) was digested to completion with the restriction endonucleases Eco RI or Hin dIII, separated by electrophoresis on a 0.9% w/v agarose gel, and transferred to Hybond-N (Amersham) by wet blotting in 20 x SSC. Filters were probed and washed as described for RNA blot analysis.
  • Soluble proteins were extracted from plant material by freezing the tissue in liquid N 2 , and grinding to a fine powder in a mortar and pestle.
  • the powdered tissue was extracted in a buffer consisting of 100mM Tris-HCl, pH 8.5, 10mM EDTA, 2mM CaCl 2 , 14 ⁇ M ⁇ -mercaptoethanol. Insoluble material was removed by centrifugation at 10,000g for 15 min. Protein concentrations were estimated by the method of Bradford (1976) with Bovine Serum Albumin (BSA) as a standard.
  • BSA Bovine Serum Albumin
  • Protein extracts and purified protein were assayed for inhibitory activity against trypsin and chymotrypsin as described by Rickauer et al . (1989). Inhibitory activity was measured against 1 pg of trypsin (TPCK-treated; Sigma) or 3 ⁇ g of chymotrypsin (TLCK-treated; Sigma).
  • Inhibitory activity of the extract was expressed as the percentage of control protease activity remaining after the protease had been pre-incubated with the extract.
  • the PI peptides from stigma, PI precursor and Asp-N processed peptides were assayed for inhibitory activity as described by Christeller et al (1989).
  • Stigmas 1000; 10g were ground to a fine powder in liquid N 2 , and extracted in buffer (100mM Tris-HCl, pH8.5, 10mM EDTA, 2mM CaCl 2 , 14 ⁇ M ⁇ -mercaptoethanol, 4ml/g tissue).
  • buffer 100mM Tris-HCl, pH8.5, 10mM EDTA, 2mM CaCl 2 , 14 ⁇ M ⁇ -mercaptoethanol, 4ml/g tissue.
  • gel filtration the inhibitory activity was precipitated with 80% w/v ammonium sulphate, the concentration required to precipitate all the proteinase inhibitory activity.
  • the ammonium sulphate pellet was resuspended in 5ml of 0.15M KCl, 10mM Tris-HCl, pH 8.1, and loaded onto a Sephadex G-50 column (2cm x 100cm) equilibrated with the same buffer.
  • the fractions (10ml) eluted from this column and containing proteinase inhibitory activity were pooled and applied to an affinity column of Chymotrypsin-Sepharose CL4B [100mg TLCK-treated ⁇ -chymotrypsin (Sigma) crosslinked to 15ml Sepharose CL4B (Pharmacia) by manufacturers instructions].
  • the column was washed with 10 volumes of 0.15M KCl/10mM Tris-HCl, pH 8.1, prior to elution of bound proteins with 7m urea, pH 3 (5 ml fractions).
  • the eluate was neutralised immediately with 200 ⁇ l 1M Tris-HCl pH 8, and dialyzed extensively against deionised H 2 O.
  • Purified PI protein was chromatographed on a reverse phase HPLC microbore column prior to automated Edman degradation on a gas phase sequencer (Mau et al ., 1986). Phenylthiohydantoin (PTH) amino acids were analysed by HPLC as described by Grego et al . (1985).
  • the purified proteinase inhibitor ( Figure 6c, lane 3) was conjugated to a carrier protein, keyhole limpet haemocyanin (KLH) (Sigma), using glutaraldehyde, as follows. 1mg of PI protein was dissolved in 1.5ml H 2 O, and mixed with 0.3 mg KLH in 0.5 ml of 0.4M phosphate buffer, pH7.5. 1ml of 20mM glutaraldehyde was added dropwise over 5 min, with stirring at room temperature. The mixture was stirred for 30 min at room temperature, 0.25ml of glycine was added, and the mixture was stirred for a further 30 min.
  • KLH keyhole limpet haemocyanin
  • the conjugated protein was then dialyzed extensively against normal saline (0.8% w/v NaCl). The equivalent of 100 ⁇ g of PI protein was used for each injection. Freund's complete adjuvant was used for the first injection, and incomplete adjuvant for two subsequent booster injections. The IgG fraction of the antiserum was separated on Protein A Sepharose (Pharmacia) according to manufacturer's instructions.
  • Protein extracts were electrophoresed in 15% w/v SDS-polyacrylamide gels (Laemmli, 1970) and transferred to nitrocellulose in 25mM Tris-HCl, 192mM glycine, 20% v/v methanol, using a BioRad Trans-Blot R Semi-dry electrophoretic transfer cell (12V, 20 min). Loading and protein transfer were checked by staining the proteins on the membranes with Ponceau S (Harlow and Lane, 1988). Membranes were blocked in 3% w/v bovine serum albumin for 1h, and incubated with the anti-PI antibody (2 ⁇ g/ml in 1% w/v BSA, Tris Buffered Saline) overnight at room temperature. Bound antibody was detected using biotinylated donkey anti-rabbit IgG (1 /500 dilution, Amersham) and the Amersham Biotin-Streptavidin system according to procedures recommended by the manufacturer.
  • Affinity-purified PI precursor (1.25mg) was incubated at 37°C with endoproteinase Asp-N (2 ⁇ g) in 100mM NH 4 HCO 3 , pH 8.5 in a total volume of 1ml for 48h. Reaction products were separated by reversed-phase HPLC using an analytical Brownlee RP-300 Aquapore column (C8, 7 ⁇ m, 4.6x100mm).
  • the column was equilibrated in 0.1% v/v TFA and peptides were eluted with the following program: 0-25%B (60% v/v acetonitrile in 0.089% v/v TFA) applied over 5min, followed by a gradient of 25-42%B over the next 40min, and ending with a gradient of 42-100%B over 5 minutes.
  • the flow rate was 1.0ml/min and peptides were detected by absorbance at 215nm. Each peak was collected manually and freeze dried. Concentration was estimated by response obtained with each peak on the UV detector at 215nm.
  • a cDNA library prepared from mRNA isolated from the stigmas and styles of mature flowers of N. alata , was screened for clones of highly expressed genes which were not associated with self-incompatibility genotype. Clones encoding a protein with some sequence identity to the type II proteinase inhibitors from potato and tomato (Thornburg et al ., 1987; Graham et al ., 1985) were selected. The largest clone, NA-PI-2, is 1360 base pairs long with an open reading frame of 1191 nucleotides. The nucleic acid sequence (SEQ ID NO. 2) and the predicted amino acid sequence (SEQ ID NO. 3) of the N. alata clone, NA-PI-2 is shown in Figure 1. There are no potential N-glycosylation sites.
  • the N. alata cDNA clone encodes a protein with six repeated domains that have high, but not perfect, sequence identity ( Figure 1). Each of these domains contains a potential reactive site which is highlighted in Figure 1.
  • the residues at the putative reactives sites of the N. alata PI are consistent with the inhibitor having two sites which would specifically inhibit chymotrypsin (Leu5-Asn6, Leu63-Asn64) and four sites specific for trypsin (Arg121-Asn122, Arg179-Asn180, Arg237-Asn238 and Arg295-Asn296).
  • Table 1 is a comparison of the percentage amino acid identity of the six domains of the PI precursor.
  • RNA Total RNA, isolated from various tissues of N. alata , was probed with the PI cDNA clone in the RNA gel blot analyses shown in Figure 2.
  • Two hybridising messages of 1.0 and 1.4kb were present in total RNA isolated from styles (including stigmas). Only the larger message, which was predominant in this tissue, is of sufficient size to encode the cDNA clone NA-PI-2 (1.4kb). The smaller message is not detected with the cDNA probe at higher stringency.
  • An homologous message of approximately 1.4kb was also present in RNA isolated from the styles of N. tabacum and N. sylvestris ( Figure 2).
  • the cDNA clone NA-PI-2 was used as a probe on the DNA gel blot shown in Figure 4 which contained genomic DNA, digested with either Eco RI or Hin dIII.
  • Eco RI produced two hybridising fragments (11kb and 7.8kb) and Hin dIII produced three large hybridising fragments (16.6, 13.5 and 10.5 kb).
  • the N-terminal amino acid sequence DRICTNCCAG(T/K)KG (SEQ ID NO. 11; SEQ ID NO. 12, respectively) was obtained from the purified PI protein. This sequence of amino acids corresponds to six regions in the deduced sequence of the cDNA clone, starting at positions 25, 83, 141, 199, 257 and 315 in Figure 1. At position 11 of the N-terminal sequence, both threonine and lysine were detected.
  • FIG. 8 is an immunoblot containing protein extracts of stigmas from flowers at different stages of development (1cm long buds to mature flowers) probed with the anti-PI antiserum. Larger cross reacting proteins of approximately 18kD, and 42kD were detected in buds from 1cm to 5cm in length in addition to the 6kD and the 32kD protein.
  • the eluant was monitored at 215nm using a Spectral Physics forward optics scanning detector with a 6-mm pathlength U-shaped axial beam capillary flow cell (LC Packings, Netherlands). Mass spectra were acquired on a Finnigan-Mat triple quadrupole mass spectrometer (modelTSQ-700, San Jose, CA) equipped with an electrospray ionisation (ESI) source (Analytica, Branford, CT). The electrospray needle was operated in positive ion mode at a voltage differential of -4 kV. The sheath liquid was 2-methoxyethanol delivered at 1 ⁇ l/min via a syringe drive (Harvard Apparatus, South Natick, MA).
  • ESI electrospray ionisation
  • the nitrogen drying gas conditions were as follows: heater temperature, 275°C; pressure, 15 psi; flow rate, ⁇ 15stdL/min.
  • the nitrogen sheath gas was supplied at 33 psi.
  • Gaseous nitrogen was obtained from a boiling liquid nitrogen source.
  • Peptides were introduced into the ESI source at 1.0 ⁇ l/min by on-line capillary RP-HPLC as described above. Spectra were acquired scanning from m / z 400 to 2000 at a rate of 3sec. Data collection and reduction were performed on a Dec5100 computer using Finnigan BIOMASSTM software.
  • the five of the six peptides of about 6kD that were predicted to be present in the purified 6kD PI preparation have been separated from each other by reversed-phase HPLC chromatography. Four peaks were obtained (Fig. 9a) and the peptides within each peak were identified by electrospray mass spectrometry (Table 2). The peptides have been designated C1, T1, T2, T3 and T4 according to their position in the PI precursor and the presence of a chymotrypsin or trypsin reactive site (Fig 9b).
  • the first HPLC peak corresponds to the chymotrypsin inhibitor C1
  • the second peak is composed of a mixture of T2 and T3 (identical to each other) and T4 that differs from T2 and T3 by one amino-acid at position 32.
  • the third peak contains the peptide T1 and the fourth peak is composed of a mixture of T1, T2/T3 and T4 (Table 2).
  • cDNA encoding the PI precursor ( Figure 1) was inserted into the Eco R1 site of the plasmid vector pVL 1392, which is the same as pVL941 (Lucknow and Summers, 1989) except that a multiple cloning site was inserted at the BamH1 site.
  • the plasmid designated pRH11 contains the PI cDNA in the correct orientation with respect to the direction of transcription directed by the polyhedrin promoter.
  • Recombinant baculovirus was obtained by co-transfection of Spodoptera frugiperda cells with baculovirus DNA and pRH11.
  • the recombinant viruses produced by homologous recombination, were plaque purified and amplified prior to infection of insect cells for protein production. All procedures for production of recombinant baculovirus, titration of the virus and maintenance and infection of the Sf9 cells were obtained from King and Posse (1992).
  • For production of the PI precursor monolayers of Sf9 cells in large flasks (175cm 2 ) were infected at the time of confluence with an inoculum of high-titre recombinant virus at a multiplicity of infection of 5-10 pfu/cell.
  • PI precursor was purified by application to a Chymotrypsin-Sepharose affinity column as described for the 6kD PI species from stigmas.
  • PI precursor eluted from the column in 7M urea, pH3 was neutralized immediately with 1M Tris-HCl buffer pH8, dialysed extensively against Milli-Q water, concentrated 20-50 fold by ultrafiltration using a Diaflow YM10 filter and stored frozen at -20°C.
  • the cDNA clone encoding the PI precursor was engineered into a baculovuirus vector for the production of the precursor from infected insect cells.
  • the insect cells produced a 42kD protein that cross reacted with the antibodies raised to the 6kD PI peptides from stigma and bound to the chymotrypsin affinity column.
  • This 42kD protein was identical in size to the 42kD precursor produced in the immature stigmas of N.alata (Fig.11) and had the N-terminal sequence LysAlaCysThrLeuAsn (SEQ ID NO. 13) demonstrating that the signal sequence had been processed correctly by the insect cells (Fig.1).
  • the 42kD protein produced in the baculovirus expression system will now be referred to as the PI precursor.
  • the 42kD PI precursor had inhibitory activity against chymotrypsin but no inhibitory activity against trypsin (Fig.13).
  • Processing of the PI precursor by the endoproteinase AspN led to the production of stable peptides of about 6kD that were partially purified by reversed phase HPLC (Fig.12).
  • These peptides have equivalent inhibitory activity against trypsin and chymotrypsin as the 6kD peptides isolated from stigma, indicating that processing of the precursor is required to activate the trypsin inhibitory activity but not all the chymotrypsin activity.
  • AspN cleaves specifically adjacent to Aspartate residues (between Asn-1 and Asp1 in Figure 10) and has no trimming activity, the peptides produced in vitro will be similiar to those produced in stigmas except for the presence of the sequence EEKKN (SEQ ID NO. 14) at the C-terminus. This provides further evidence that precise processing of the N-and C-termini is not required to obtain an active 6kD PI peptide.
  • Asp-N1 is more efficient at inhibiting chymotrypsin than trypsin and is thus likely to be predominantly a C1 analogue (Fig.9b).
  • Asp-N2 is a more efficient trypsin inhibitor and probably contains the T1-T4 analogues.
  • Table 3 shows the inhibitory activity of the pooled 6kD PI peptides (C1, T1, T2/T3, T4), the mixture of trypsin inhibitors T2/T3 and T4, and the chymotrypsin inhibitor C1 against the proteases in the gut of various members of the Lepidoptera, Coleoptera, Orthoptera and Diptera.
  • the pooled peptides and the trypsin inhibitors had an equivalent effect against the gut proteases with the degree of inhibition ranging from 37-79% depending on the insect tested.
  • the inhibitors had negligible effect on the gut proteases of the potato tuber moth, P.opercullela .
  • the chymotrypsin inhibitor C1 also affected the activity of the proteases but was less effective than the trypsin inhibitors in five cases ( W.cervinata, L.serricata, C.zealandica, P.octo , sugar cane grub).
  • alata trypsin inhibitors peak 2 from HPLC
  • Heliothis armigera Helicoverpa armigera
  • Tobacco budworm Lepidoptera Heliothis punctigera
  • Helicoverpa punctigera Native budworm
  • Lepidoptera Teleogryllus commodus Black field cricket Orthoptera Agrotis infusa Common cutworm, adults known as the Bogong moth, Lepidoptera Wiseana cervinata Porina, native to New Zealand, Lepidoptera Lucilla sericata Green blow fly, Diptera, assayed at pH8 Chrysomya rufifacies Hairy maggot blow fly, Diptera, assayed at pH8 Aphodius tasmaniae
  • Kenyan grass grub Black-headed pasture cockchafer, Coleoptera Costelytra zealandica New Zealand grass grub, Coleoptera Spodoptera litura Tropical armyworm, Lepidoptera Phthorimaea operculle

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